Release Channel Of Sarcoplasmic Reticulum Research Articles

Abstract 14182: A Missense Mutation in Ryanodine Receptor 2 Leads to Structural Remodeling of the Heart and Cardiac Arrhythmia in a Rabbit Model

Ryanodine receptor 2 (RyR2) is the Ca 2+ release channel of sarcoplasmic reticulum that provides the majority of Ca 2+ necessary for contractions of the heart. RyR2 mutations are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited arrhythmogenic disorder with normal heart structure. There are reports suggesting that RyR2 mutations can also lead to structural heart disease, notably arrhythmogenic right ventricular cardiomyopathy (ARVC); however, additional research is required to support this association. We generated a rabbit model carrying the missense mutation RyR2-V2475F, linked to CPVT in humans. Heterozygous (V2475F +/- ) rabbits show a CPVT-like syndrome and display arrhythmia under pharmacological stress. Homozygotes (V2475F +/+ ) show a stronger phenotype, with 37% of the animals (13/35) experiencing sudden death at 22.5±3.8 weeks old during regular housing. Remarkably, gross examination of V2475F +/+ hearts showed significant structural remodeling, characterized by fibrous infiltrations and thinning of the right ventricle with or without left ventricle involvement. This phenotype is reminiscent of ARVC. Lead II ECG obtained in conscious rabbits by telemetry showed that V2475F +/+ animals have significant bradycardia (144.6±11.1 bpm) with prolonged QT interval (QTc: 180.4±8.2 ms, n=6) compared to WT (HR: 224.9±4.2 bpm, p=0.04; QTc: 157.4±5.3 ms, p=0.02, n=5). After subcutaneous injection of 0.5 mg/kg of isoproterenol, WT rabbits showed the expected positive chronotropic response (HR, 367±10 bpm, n=3) while V2475F +/+ animals developed bouts of ventricular tachycardia. Mild emotional stress applied by transporting the animal around the laboratory, also induced ventricular arrhythmia in 100% (5/5) V2475F +/+ rabbits, while WT littermates were unaffected. RyR2-V2475F expressed in a rabbit model triggers remarkably different phenotypes based on the zygosity of the animals, ranging from a purely arrhythmogenic syndrome (CPVT) in V2475F +/- animals to severe structural disease compatible with ARVC in V2475F +/+ rabbits. The RyR2-V2475F rabbit model is therefore an excellent model to dissect the intersection of arrhythmic and structural phenotypes due to RyR2 dysfunction.

The Efficacy of Microcurrent Therapy on Eccentric Contraction-Induced Muscle Damage in Rat Fast-Twitch Skeletal Muscle

Microcurrent (MC) therapy, in which a very small electric current is applied to the body, has widely been used to promote tissue healing and relieve symptoms. The aim of this study was to examine the effect of MC treatment on eccentric contraction (ECC)-induced muscle damage in rat fast-twitch skeletal muscles. Tibialis anterior muscles underwent 200 repeated ECCs in situ and were then stimulated (25 μA, 0.3 Hz) for 20 min (MC treatment). MC treatment was performed immediately after ECC and during a recovery period of 3 days (a total of 4 times). Three days after ECC, the muscles were excised and used for measure of force output and for biochemical analyses. In MC-treated muscles, tetanic forces at 20 Hz and 100 Hz were partially and fully restored, respectively, whereas in non-treated muscles, both forces remained depressed. Biochemical analyses revealed that MC treatment partially or completely inhibited ECC-induced reductions: in 1) the Ca2+-release function of sarcoplasmic reticulum (SR), 2) proteolysis of ryanodine receptor, a Ca2+ release channel of SR, and 3) myosin ATPase activity. On the other hand, MC treatment was unable to lessen increases in the activity of calpain, a cytosolic, Ca2+-activated neutral protease. These results indicate that MC treatment results in beneficial effects, such as restoration of muscle performance following ECC, although the precise mechanisms are still unknown at this time.

Arrhythmogenic mechanisms in ryanodine receptor channelopathies.

Ryanodine receptors (RyRs) are the calcium release channels of sarcoplasmic reticulum (SR) that provide the majority of cal-cium ions (Ca2+) necessary to induce contraction of cardiac and skeletal muscle cells. In their intracellular environment, RyR channels are regulated by a variety of cytosolic and luminal factors so that their output signal (Ca2+) induces finely-graded cell contraction without igniting cellular processes that may lead to aberrant electrical activity (ventricular arrhythmias) or cellular remodeling. The importance of RyR dysfunction has been recently highlighted with the demonstration that point mutations in RYR2, the gene encoding for the cardiac isoform of the RyR (RyR2), are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), an arrhythmogenic syndrome characterized by the development of adrenergically-mediated ventricular tachycardia in individuals with an apparently normal heart. Here we summarize the state of the field in regards to the main arrhythmogenic mechanisms triggered by RyR2 channels harboring mutations linked to CPVT. Most CPVT mutations characterized to date endow RyR2 channels with a gain of function, resulting in hyperactive channels that release Ca2+ spontaneously, especially during diastole. The spontaneous Ca2+ release is extruded by the electrogenic Na+/Ca2+ exchanger, which depolarizes the external membrane (delayed afterdepolarization or DAD) and may trigger untimely action potentials. However, a rare set of CPVT mutations yield RyR2 channels that are intrinsically hypo-active and hypo-responsive to stimuli, and it is unclear whether these channels release Ca2+ spontaneously during diastole. We discuss novel cellular mechanisms that appear more suitable to explain ventricular arrhythmias due to RyR2 loss-of-function mutations.

Malignant Hyperthermia: Clinical and Molecular Aspects

Malignant hyperthermia (MH) is a potentially lethal pharmacogenetic disorder that affects genetically predisposed individuals. It manifests in susceptible individuals in response to exposure to Inhalant anesthetics, depolarizing muscle relaxants or extreme physical activity in hot environments. During exposure to these triggering agents, there is a rapid and sustained increase of myoplasmic calcium (Ca(2+)) concentration induced by hyperactivation of ryanodine receptor of skeletal muscle (RyR1), causing a profound change in Ca(2+) homeostasis, featuring a hypermetabolic state. RyR1, Ca(2+) release channels of sarcoplasmic reticulum, is the primary locus for MH susceptibility. Several mutations in the gene encoding the protein RyR1 have been identified; however, other genes may be involved. Actually, the standard method for diagnosing MH susceptibility is the muscle contracture test for exposure to halothane-caffeine (CHCT) and the only treatment is the use of dantrolene. However, with advances in molecular genetics, a full understanding of the disease etiology may be provided, favoring the development of an accurate diagnosis, less invasive, with DNA test, and also will provide the development of new therapeutic strategies for treatment of MH. Thus, this brief review aims to integrate molecular and clinical aspects of MH, gathering input for a better understanding of this channelopathy.

Arrhythmogenic Ion-Channel Remodeling in Heart Failure and Upstream Approach

Class I antiarrhythmic drugs with Na+ channel blocking action cannot be readily used for the treatment of various arrhythmias associated with heart failure because the antiarrhthmic drugs may worsen cardiac function. In failing hearts ion-channel remodeling, such as downregulation of K channels and connexin 43, upregulation of Na-Ca exchanger and HCN channels, and instability of Ca release channels in sarcoplasmic reticulum, can be observed. These electrophysiological abnormalities can produce severe ventricular tachyarrhythmias through reentrant and automatic mechanisms. Neurohumoral factors such as renin-angiotensin system, endothelin system and sympathetic nervous system play an important role in the establishment of the ion-channel remodeling. Upstream approach with blockade of the neurohumoral factors may prevent lethal ventricular arrhythmias. In congestive heart failure atrial tissues are expanded and susceptibility to atrial fibrillation is increased. Control of neurohumoral factors may be also beneficial for the prevention of atrial fibrillation. Recently it has been suggested that inflammatory mechanisms may be also involved in not only the deterioration of heart failure but also induction of atrial fibrillation and ventricular tachyarrhythmias. Drugs with anti-inflammatory action, including the antiarrhythmic drug amiodarone, may be effective for the treatment of arrhythmias associated with heart failure. Current perspective of upstream approach to preventing atrial and ventricular arrhythmias associated with heart failure will be discussed.

Targeting intracellular calcium cycling in catecholaminergic polymorphic ventricular tachycardia: a theoretical investigation

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a malignant arrhythmogenic disorder linked to mutations in the cardiac ryanodine receptor (RyR2) and calsequestrin, predisposing the young to syncope and cardiac arrest. To define the role of β-adrenergic stimulation (BAS) and to identify potential therapeutic targeted sites relating to intracellular calcium cycling, we used a Luo-Rudy dynamic ventricular myocyte model incorporated with interacting Markov models of the L-type Ca(2+) channel (I(Ca,L)) and RyR2 to simulate the heterozygous state of mouse RyR2 R4496C mutation (RyR2(R4496C+/-)) comparable with CPVT patients with RyR2 R4497C mutation. Characteristically, in simulated cells, pacing at 4 Hz or faster or pacing at 2 Hz under BAS with effects equivalent to those of isoproterenol at ≥ 0.1 μM could readily induce delayed afterdepolarizations (DADs) and DAD-mediated triggered activity (TA) in RyR2(R4496C+/-) but not in the wild-type via enhancing both I(Ca,L) and sarcoplasmic reticulum (SR) Ca(2+) ATPase (I(UP)). Moreover, with the use of steady state values of isolated endocardial (Endo), mid-myocardial (M), and epicardial (Epi) cells as initial data for conducting single cell and one-dimensional strand studies, the M cell was more vulnerable for developing DADs and DAD-mediated TA than Endo and Epi cells, and the gap junction coupling represented by diffusion coefficient (D) of ≤ 0.000766*98 cm(2)/ms was required for generating DAD-mediated TA in RyR2(R4496C+/-). Whereas individual reduction of Ca(2+) release channel of SR and Na-Ca exchanger up to 50% was ineffective, 30% or more reduction of either I(Ca,L) or I(UP) could totally suppress the inducibility of arrhythmia under BAS. Of note, 15% reduction of both I(Ca,L) and I(UP) exerted a synergistic antiarrhythmic efficacy. Findings of this model study confirm that BAS facilitates induction of ventricular tachyarrhythmias via its action on intracellular Ca(2+) cycling and a pharmacological regimen capable of reducing I(Ca,L) could be an effective adjunctive to β-adrenergic blockers for suppressing ventricular tachyarrhythmias during CPVT.

Predicting Local SR Ca2+ Dynamics during Ca2+ Wave Propagation in Ventricular Myocytes

Of the many ongoing controversies regarding the workings of the sarcoplasmic reticulum (SR) in cardiac myocytes, two unresolved and interconnected topics are 1), mechanisms of calcium (Ca2+) wave propagation, and 2), speed of Ca2+ diffusion within the SR. Ca2+ waves are initiated when a spontaneous local SR Ca2+ release event triggers additional release from neighboring clusters of SR release channels (ryanodine receptors (RyRs)). A lack of consensus regarding the effective Ca2+ diffusion constant in the SR (DCa,SR) severely complicates our understanding of whether dynamic local changes in SR [Ca2+] can influence wave propagation. To address this problem, we have implemented a computational model of cytosolic and SR [Ca2+] during Ca2+ waves. Simulations have investigated how dynamic local changes in SR [Ca2+] are influenced by 1), DCa,SR; 2), the distance between RyR clusters; 3), partial inhibition or stimulation of SR Ca2+ pumps; 4), SR Ca2+ pump dependence on cytosolic [Ca2+]; and 5), the rate of transfer between network and junctional SR. Of these factors, DCa,SR is the primary determinant of how release from one RyR cluster alters SR [Ca2+] in nearby regions. Specifically, our results show that local increases in SR [Ca2+] ahead of the wave can potentially facilitate Ca2+ wave propagation, but only if SR diffusion is relatively slow. These simulations help to delineate what changes in [Ca2+] are possible during SR Ca2+release, and they broaden our understanding of the regulatory role played by dynamic changes in [Ca2+]SR.

Competitive Regulation of Calcium and Zinc Ions in Cardiomyocyte Contraction-Relaxation Function

Zinc (Zn2+) and calcium (Ca2+) ions are divalent cations having common chemical properties leading to their competing for the same regulatory channels and pumps in the intact cardiomyocyte. Diastolic dysfunction may be due in part to elevated diastolic Ca2+ concentration ([Ca2+]). We hypothesized that Zn2+ reduces systolic and enhances diastolic function due to its effects on Ca2+ regulation. We examined the effects of 32µM extracellular zinc ([Zn2+]ext) exposure and intracellular zinc ([Zn2+]int) accumulation on rat cardiomyocyte function. We measured sarcomere dynamics, [Ca2+]int by Fura-2FF and [Zn2+]int by FluoZin-3 under three [Zn2+] conditions: no [Zn2+]ext and low [Zn2+]int; 32µM [Zn2+]ext and low [Zn2+]int; 32µM [Zn2+]ext and high [Zn2+]int. Cardiomyocytes were paced at 2Hz, exposed to 2mM [Ca2+]ext at 37°C. After reaching [Zn2+]int steady-state, 10mM caffeine was rapidly applied to measure sarcoplasmic reticulum (SR) Ca2+ content and Na+-Ca2+ exchanger (NaCaX) efflux rate. Sarcomere shortening velocity and peak shortening were significantly (P<0.05) reduced with [Zn2+]ext exposure in either low or high [Zn2+]int conditions. Interestingly, peak shortening was significantly enhanced with high [Zn2+]int compared to low [Zn2+]int. Diastolic sarcomere length was significantly increased with high [Zn2+]int. Peak [Ca2+]int was significantly reduced under 32μM [Zn2+]ext with high [Zn2+]int, which was consistent with lower SR Ca2+ content detected by caffeine experiment. SR Ca2+ uptake rate by SERCA and NaCaX efflux rate were not affected by [Zn2+]int. All the above changes due to Zn2+ were not observed in control cardiomyocytes without [Zn2+]ext exposure. These findings suggest that Zn2+ competes with Ca2+ for calcium channels (L-type and SR release channels) and thereby reduces contractile function without affecting SERCA or NaCaX. Interestingly, high [Zn2+]int causes a slightly increased contractile function despite the reduction in peak [Ca2+]int, suggesting that [Zn2+]int enhances myofilament contraction by mechanisms yet to be explained.

Caffeine administration results in greater tension development in previously fatigued canine muscle in situ

In isolated single skeletal myocytes undergoing long-term fatiguing contractions, caffeine (CAF) can result in nearly immediate restoration of generated tension to near-prefatigue levels by increasing Ca2+ release via activation of sarcoplasmic reticulum release channels. This study tested whether arterial CAF infusion (>5 mm) would cause a similar rapid restoration of tetanic isometric tension during contractions to fatigue in perfused canine hindlimb muscle in situ. Tetanic contractions were elicited by electrical stimulation (200 ms trains, 50 Hz, 1 contraction s(-1)), and biopsies were taken from the muscle at rest and during contractions: (1) following the onset of fatigue (tension approximately 60% of initial value); and (2) following CAF administration. Resting muscle ATP, PCr and lactate contents were 25.2 +/- 0.4, 76.9 +/- 3.3 and 14.4 +/- 3.3 mmol (kg dry weight)(-1), respectively. At fatigue, generated tetanic tension was 61.1 +/- 6.9% of initial contractions. There was a small but statistically significant recovery of tetanic tension (64.9 +/- 6.6% of initial value) with CAF infusion, after which the muscle showed incomplete relaxation. At fatigue, muscle ATP and PCr contents had fallen significantly (P < 0.05) to 18.1 +/- 1.1 and 18.9 +/- 2.1 mmol (kg dry weight)(-1), respectively, and lactate content had increased significantly to 27.7 +/- 5.4 mmol (kg dry weight)(-1). Following CAF, skeletal muscle ATP and PCr contents were significantly lower than corresponding fatigue values (15.0 +/- 1.3 and 10.9 +/- 2.2 mmol (kg dry weight)(-1), respectively), while lactate was unchanged (22.2 +/- 3.9 mmol (kg dry weight)(-1)). These results demonstrate that caffeine can result in a small, but statistically significant, recovery of isometric tension in fatigued canine hindlimb muscle in situ, although not nearly to the same degree as seen in isolated single muscle fibres. This suggests that, in this in situ isolated whole muscle model, alteration of Ca2+ metabolism is probably only one cause of fatigue.

Antisense strategies for treatment of heart failure.

AbstractIn the mammalian heart, intracellular Ca2+ movements are tightly regulated at various levels within the cell (1). The sarcoplasmic reticulum (SR) plays an important role in orchestrating the movement of calcium during each contraction and relaxation. Excitation leads to the opening of voltage-gated L-type Ca2+ channels, allowing the entry of a small amount of Ca2+ into the cell. Through a coupling mechanism between the L-type Ca2+ channel and the SR release channels (ryanodine receptors), a larger amount of Ca2+ is released, activating the myofilaments leading to contraction. During relaxation, Ca2+ is reaccumulated back into the SR by the SR Ca2+ adenosine triphosphatase (ATPase) pump (SERCA2a) and extruded extracellularly by the sarcolemmal Na+/Ca2+ exchanger. The contribution of each of these mechanisms for lowering cytosolic Ca2+ varies with species. In humans, approx 70% of the Ca2+ is removed by SERCA2a and approx 30% by the Na+/Ca2+ exchanger (1). The Ca2+ pumping activity of SERCA2a is influenced by phospholamban. In the unphosphorylated state, phospholamban inhibits the Ca2+-ATPase, whereas phosphorylation of phospholamban by cyclic adenosine monophosphate (cAMP)-dependent protein kinase and by Ca2+-calmodulin-dependent protein kinase reverses this inhibition (2). Ca2+ transients recorded from failing human myocardial cells or trabeculae reveal a significantly prolonged Ca2+ transient with an elevated end-diastolic intracellular Ca2+ (3,4).KeywordsSarcoplasmic ReticulumSarcoplasmic Reticulum CalciumHuman CardiomyocytesAdenoviral Gene TransferAntisense StrategyThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Ryanodine receptor channelopathies

Ryanodine receptors (RyR) are the Ca 2+ release channels of sarcoplasmic reticulum that provide the majority of the [Ca 2+] necessary to induce contraction of cardiac and skeletal muscle cells. In their cellular environment, RyRs are exquisitely regulated by a variety of cytosolic factors and accessory proteins so that their output signal (Ca 2+) induces cell contraction without igniting signaling pathways that eventually lead to contractile dysfunction or pathological cellular remodeling. Here we review how dysfunction of RyRs, most commonly expressed as enhanced Ca 2+ release at rest (skeletal muscle) or during diastole (cardiac muscle), appears to be the fundamental mechanism underlying several genetic or acquired syndromes. In skeletal muscle, malignant hyperthermia and central core disease result from point mutations in RYR1, the skeletal isoform of RyRs. In cardiac muscle, RYR2 mutations lead to catecholaminergic polymorphic ventricular tachycardia and other cardiac arrhythmias. Lastly, an altered phosphorylation of the RyR2 protein may be involved in some forms of congestive heart failure.

Role of Ryanodine Receptors in Chloroform-Induced Contraction in Swine Tracheal Smooth Muscle

Chloroform has been reported to induce inhalation intoxication in the respiratory tract. The purpose of this study was to investigate the effects and mechanisms of chloroform on muscle contraction in isolated swine tracheal smooth muscle. Chloroform (30–1000 ppm) reversibly and concentration-dependently provoked smooth muscle contraction. Muscarinic and α-adrenergic receptor antagonists did not alter chloroform-induced muscle contraction, indicating muscarinic and α-adrenergic stimulation may not be involved in chloroform-induced responses. Caffeine (10 mM) was observed to directly evoke tracheal smooth muscle contraction, but ryanodine (1 μM) was not. However, ryanodine and caffeine abolished chloroform-induced smooth muscle contraction by 80.0 ± 8.0 and 79.6 ± 6.0%, respectively. Caffeine combined with ryanodine completely blocked chloroform-induced contractile responses. Thus, it suggests that chloroform released Ca 2+ from ryanodine-sensitive internal Ca 2+ pools. Although short-term removal of Ca 2+ from extracellular environment slightly decreased chloroform-induced contractile responses, L-type Ca 2+ channel blockers did not alter tracheal smooth muscle contraction induced by chloroform. Collectively, our results indicated that chloroform directly and concentration-dependently provoked muscle contraction in swine tracheal smooth muscle, which may result from the activation of ryanodine receptor Ca 2+ release channel in sarcoplasmic reticulum but may not depend on muscarinic and adrenergic activation and Ca 2+ entry from the extracellular environment.

Role of Ryanodine Receptors in Chloroform-Induced Contraction in Swine Tracheal Smooth Muscle

Chloroform has been reported to induce inhalation intoxication in the respiratory tract. The purpose of this study was to investigate the effects and mechanisms of chloroform on muscle contraction in isolated swine tracheal smooth muscle. Chloroform (30–1000 ppm) reversibly and concentration-dependently provoked smooth muscle contraction. Muscarinic and α-adrenergic receptor antagonists did not alter chloroform-induced muscle contraction, indicating muscarinic and α-adrenergic stimulation may not be involved in chloroform-induced responses. Caffeine (10 mM) was observed to directly evoke tracheal smooth muscle contraction, but ryanodine (1 μM) was not. However, ryanodine and caffeine abolished chloroform-induced smooth muscle contraction by 80.0 ± 8.0 and 79.6 ± 6.0%, respectively. Caffeine combined with ryanodine completely blocked chloroform-induced contractile responses. Thus, it suggests that chloroform released Ca 2+ from ryanodine-sensitive internal Ca 2+ pools. Although short-term removal of Ca 2+ from extracellular environment slightly decreased chloroform-induced contractile responses, L-type Ca 2+ channel blockers did not alter tracheal smooth muscle contraction induced by chloroform. Collectively, our results indicated that chloroform directly and concentration-dependently provoked muscle contraction in swine tracheal smooth muscle, which may result from the activation of ryanodine receptor Ca 2+ release channel in sarcoplasmic reticulum but may not depend on muscarinic and adrenergic activation and Ca 2+ entry from the extracellular environment.

Identification of a Dantrolene-binding Sequence on the Skeletal Muscle Ryanodine Receptor

Dantrolene is a drug that suppresses intracellular Ca(2+) release from sarcoplasmic reticulum (SR) in skeletal muscle and is used as a therapeutic agent in individuals susceptible to malignant hyperthermia. Although its precise mechanism of action has not been elucidated, we have identified the N-terminal region (amino acids 1-1400) of the skeletal muscle isoform of the ryanodine receptor (RyR1), the primary Ca(2+) release channel in SR, as a molecular target for dantrolene using the photoaffinity analog [(3)H]azidodantrolene. Here, we demonstrate that heterologously expressed RyR1 retains its capacity to be specifically labeled with [(3)H]azidodantrolene, indicating that muscle specific factors are not required for this ligand-receptor interaction. Synthetic domain peptides of RyR1 previously shown to affect RyR1 function in vitro and in vivo were exploited as potential drug binding site mimics and used in photoaffinity labeling experiments. Only DP1 and DP1-2s, peptides containing the amino acid sequence corresponding to RyR1 residues 590-609, were specifically labeled by [(3)H]azidodantrolene. A monoclonal anti-RyR1 antibody that recognizes RyR1 and its 1400-amino acid N-terminal fragment recognizes DP1 and DP1-2s in both Western blots and immunoprecipitation assays and specifically inhibits [(3)H]azidodantrolene photolabeling of RyR1 and its N-terminal fragment in SR. Our results indicate that synthetic domain peptides can mimic a native, ligand-binding conformation in vitro and that the dantrolene-binding site and the epitope for the monoclonal antibody on RyR1 are equivalent and composed of amino acids 590-609.

Doxorubicin and C-13 Deoxydoxorubicin Effects on Ryanodine Receptor Gene Expression

Chronic anthracycline administration to rabbits causes impairment of cardiac contractility and decreased gene expression of the calcium-induced calcium release channel of sarcoplasmic reticulum (SR), the ryanodine receptor (RYR2). The C-13 hydroxy metabolite (doxorubicinol), formed in the heart, has been hypothesized to contribute to anthracycline cardiotoxicity. C-13 deoxydoxorubicin is an analog unable to form the C-13 hydroxy metabolite. Therefore, doxorubicin, C-13 deoxydoxorubicin, or saline was administered to rabbits (1 mg/kg iv twice weekly for 8 weeks). Left ventricular fractional shortening (LVFS) was decreased by chronic treatment with doxorubicin (28 ± 2%; P < 0.05), but not C-13 deoxydoxorubicin (33 ± 2%) compared to age-matched pair-fed controls. Doxorubicin, but not C-13 deoxydoxorubicin, caused a significant reduction (P < 0.02) in the ratio of RYR2/Ca-Mg ATPase (SERCA2) mRNA levels (0.57 ± 0.1 vs 1.22 ± 0.2, respectively) in the left ventricle. This suggests that doxorubicinol may contribute to the downregulation of cardiac RYR2 expression in chronic doxorubicin cardiotoxicity.

Progression of Heart Failure

The cardiac ryanodine receptor (RyR2)/Ca2+ release channel on the sarcoplasmic reticulum (SR) is regulated by evolutionarily highly conserved signaling pathways that control excitation-contraction (EC) coupling in the heart. Phosphorylation of RyR2 by cAMP-dependent protein kinase (PKA) plays a key role in regulating the channel in response to stress via activation of the sympathetic nervous system (the “fight-or-flight response”).1 Maladaptive PKA hyperphosphorylation of RyR2 in failing hearts alters channel function, which may cause depletion of SR Ca2+ and diastolic release of SR Ca2+. This can initiate delayed afterdepolarizations that trigger ventricular arrhythmias.1 Mutations in RyR2 recently have been identified in patients with catecholaminergic induced sudden cardiac death (SCD).2–4⇓⇓ There may be a direct link between the PKA hyperphosphorylation of RyR2 that occurs during the progression of heart failure and fatal cardiac arrhythmias. Regulation of cardiac EC coupling by the release of Ca2+ from the SR via RyR2 in cardiomyocytes, known as Ca2+-induced Ca2+ release (CICR), has been appreciated for more than a decade.5,6⇓ Furthermore, it is well known that the amplitude of the Ca2+ transient generated by SR Ca2+ release determines contractile force in cardiomyocytes. The systems that regulate SR Ca2+ release include: (1) the triggers (predominantly Ca2+ influx through the voltage-gated Ca2+ channel on the plasma membrane); (2) the SR Ca2+ release channel or type 2 RyR2; and (3) the SR Ca2+ reuptake pump (SERCA2a) and its regulator phospholamban. These systems (trigger, release, and reuptake) are modulated by signaling pathways, including the β-adrenergic receptor (β-AR) signaling pathway (ie, phosphorylation by PKA). Activation of the sympathetic nervous system in response to stress results in elevation of cAMP levels and activation of PKA. Phosphorylation of RyR2 may not correlate directly …

CADP-ribose activates reconstituted ryanodine receptors from coronary arterial smooth muscle.

The present study was designed to test the hypothesis that cADP-ribose (cADPR) increases Ca(2+) release through activation of ryanodine receptors (RYR) on the sarcoplasmic reticulum (SR) in coronary arterial smooth muscle cells (CASMCs). We reconstituted RYR from the SR of CASMCs into planar lipid bilayers and examined the effect of cADPR on the activity of these Ca(2+) release channels. In a symmetrical cesium methanesulfonate configuration, a 245 pS Cs(+) current was recorded. This current was characterized by the formation of a subconductance and increase in the open probability (NP(o)) of the channels in the presence of ryanodine (0.01-1 microM) and imperatoxin A (100 nM). A high concentration of ryanodine (50 microM) and ruthenium red (40-80 microM) substantially inhibited the activity of RYR/Ca(2+) release channels. Caffeine (0.5-5 mM) markedly increased the NP(o) of these Ca(2+) release channels of the SR, but D-myo-inositol 1,4,5-trisphospate and heparin were without effect. Cyclic ADPR significantly increased the NP(o) of these Ca(2+) release channels of SR in a concentration-dependent manner. Addition of cADPR (0.01 microM) into the cis bath solution produced a 2.9-fold increase in the NP(o) of these RYR/Ca(2+) release channels. An eightfold increase in the NP(o) of the RYR/Ca(2+) release channels (0.0056 +/- 0.001 vs. 0.048 +/- 0.017) was observed at a concentration of cADPR of 1 microM. The effect of cADPR was completely abolished by ryanodine (50 microM). In the presence of cADPR, Ca(2+)-induced activation of these channels was markedly enhanced. These results provide evidence that cADPR activates RYR/Ca(2+) release channels on the SR of CASMCs. It is concluded that cADPR stimulates Ca(2+) release through the activation of RYRs on the SR of these smooth mucle cells.

Effects of cartap on isolated mouse phrenic nerve diaphragm and its related mechanism.

Cartap, a nereistoxin analogue pesticide, is reported to have no irritation to eyes in rabbits. However, we have demonstrated recently that cartap could actually cause acute death in rabbits via ocular exposure. Our preliminary study with isolated mouse phrenic nerve diaphragms has shown that instead of neuromuscular blockade, cartap caused muscular contracture. The objective of the study was to examine the effect of cartap on the neuromuscular junction in more detail and to investigate its possible underlying mechanism with isolated mouse phrenic nerve diaphragms and sarcoplasmic reticulum (SR) vesicles. Cartap or nereistoxin at various concentrations was added in the organ bath with isolated mouse phrenic nerve diaphragm and both nerve- and muscle-evoked twitches were recorded. Instead of blocking the neuromuscular transmission as nereistoxin did, cartap caused contracture in stimulated or quiescent isolated mouse phrenic nerve diaphragm. Both the cartap-induced muscular contracture force and the time interval to initiate the contracture were dose-dependent. The contracture induced by cartap was not affected by the pretreatment of the diaphragm with the acetylcholine receptor blocker alpha-bungarotoxin; the Na(+) channel blocker tetrodotoxin; or various Ca(2+) channel blockers, NiCl(2), verapamil, and nifedipine. On the contrary, the contracture was significantly inhibited when the diaphragm was pretreated with ryanodine or EGTA containing Ca(2+)-free Krebs solution or in combination. This suggested that both internal and extracellular Ca(2+) might participate in cartap-induced skeletal muscle contracture. Moreover, cartap inhibited the [(3)H]-ryanodine binding to the Ca(2+) release channel of SR in a dose-dependent manner. Additionally, cartap could induce a significant reduction in Ca(2+)-ATPase activity of SR vesicles at a relatively high dose. The results suggested that cartap might cause the influx of extracellular Ca(2+) and the release of internal Ca(2+), with subsequent induction of muscular contracture in the isolated mouse phrenic nerve diaphragm. Based on these findings, we propose that the acute death of rabbits following ocular exposure to cartap might have resulted from respiratory failure secondary to diaphragm contracture.

Modulation of the Ca 2+ release channel of sarcoplasmic reticulum by amiloride analogs

Dichlorobenzamil, phenamil and other amiloride analogs (1–100 μM) elicit transient tension in rabbit skinned muscle fibers. Tension requires preloading of Ca 2+ into the sarcoplasmic reticulum, is facilitated by low-[Mg 2+] solutions, abolished by ruthenium red or by functional disruption of the sarcoplasmic reticulum, and is followed by inhibition of the caffeine-evoked tension. Bilayer recording of Cs + currents through the sarcoplasmic reticulum Ca 2+ release channel reveals that phenamil (10–100 μM) increases the open channel probability, whereas dichlorobenzamil affects the channel activity in a complex concentration- and time-dependent manner: stimulation occurs throughout exposure to 10 μM, but is followed by channel blockade when 100 μM dichlorobenzamil is used. It is concluded that stimulation of the sarcoplasmic reticulum Ca 2+ release channel accounts for the dichlorobenzamil- or phenamil-induced tension in skinned fibers, whereas depletion of sarcoplasmic reticulum Ca 2+ stores and channel block (with dichlorobenzamil) explains the inhibition of the caffeine-evoked tension by amiloride analogs.

Constitutive beta2-adrenergic signalling enhances sarcoplasmic reticulum Ca2+ cycling to augment contraction in mouse heart.

1. Transgenic overexpression of the beta2-adrenergic receptor (beta2AR) in mouse heart augments baseline cardiac function in a ligand-independent manner, due to the presence of spontaneously active beta2AR (beta2AR*). This study aims to elucidate the mechanism of beta2AR*-mediated modulation of cardiac excitation-contraction (EC) coupling. 2. Confocal imaging was used to analyse Ca2+ sparks and spatially resolve Ca2+ transients in single ventricular myocytes from transgenic (TG4) and non-transgenic (NTG) littermates. Whole-cell voltage- and current-clamp techniques were used to record L-type Ca2+ currents (ICa) and action potentials, respectively. 3. In the absence of any beta2AR ligand, TG4 myocytes had greater contraction amplitudes, larger Ca2+ transients and faster relaxation times than did NTG cells. 4. The action potentials of TG4 and NTG myocytes were similar, except for a prolonged end-stage repolarization in TG4 cells; the ICa density and kinetics were nearly identical. The relationship between peak Ca2+ and contraction, which reflects myofilament Ca2+ sensitivity, was similar. 5. In TG4 cells, the frequency of Ca2+ sparks (spontaneous or evoked at -40 mV) was 2-7 times greater, despite the absence of change in the resting Ca2+, sarcoplasmic reticulum (SR) Ca2+ content, and ICa. Individual sparks were brighter, broader and lasted longer, leading to a 2.3-fold greater signal mass. Thus, changes in both spark frequency and size underlie the greater Ca2+ transient in TG4 cells. 6. The inverse agonist ICI 118,551 (ICI, 5 x 10-7 M), which blocks spontaneous beta2AR activation, reversed the aforementioned beta2AR* effects on cardiac EC coupling without affecting the sarcolemmal ICa. However, ICI failed to detect significant constitutive beta2AR activity in NTG cells. 7. We conclude that beta2AR*-mediated signalling enhances SR release channel activity and Ca2+-induced Ca2+ release in TG4 cardiac myocytes, and that beta2AR* enhances EC coupling by reinforcing SR Ca2+ cycling (release and reuptake), but bypassing the sarcolemmal ICa.